Stem Rust Resistance and Resistance-Associated Genes in 64 Wheat Cultivars from Southern Huanghuai, China

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease that affects wheat globally. The planting of resistant cultivars is the most cost-effective strategy for controlling this disease. The Huanghuai region, as a major wheat-growing area, plays a crucial role in the spread and prevalence of wheat stem rust in China. In this study, 64 wheat accessions from this region were tested at the adult stage against two major Pgt races, 34MKGQM and 21C3CTHQM. DNA markers associated with the known resistance genes Sr31, Sr24, Sr25, Sr26, and Sr38 were measured to determine their presence in the tested accessions. In the 2023 field tests, 5 (7.8%) accessions were immune to 21C3CTHQM and 34MKGQM, while 35 (54.7%) and 39 (60.9%) were moderately resistant and resistant, respectively. The remaining 20 (30.7%) accessions were moderately susceptible and susceptible. In the 2024 tests, 12 (18.8%) and 14 (21.9%) entries were immune to both races; 29 (45.3%) and 30 (46.9%) were moderately resistant and resistant, respectively. Only two cultivars, Xinong 816 and Yimai 211, were immune in both years, and three entries showed some degrees of resistance in both years. Seven cultivars, including Zhongzhimai 23, Longxing 1, Yunong 937, Huaguan 301, Wanke 800, Shaanhe 285, and Yunong 612, showed increased susceptibility. DNA markers showed that 30 entries carried Sr31, while 6 entries carried Sr38. Genes Sr24, Sr25, and Sr26, which confer good resistance to the globally prevalent cultivars TKTTF and TTTRF, were absent from the set of tested entries. While this study surveyed the resistance levels of a cross-section of wheat from the southern part of the Huanghuai region and confirmed the presence of two known resistance genes, the basis of immunity or high levels of resistance in several lines remains obscure.


Introduction
Wheat is one of the world's most important food crops and is widely cultivated almost around the world, with a range of cultivars possessing different characteristics [1].Wheat cultivation and yield are crucial for maintaining national food security, promoting economic development, and improving living standards.Wheat stem rust caused by Puccinia graminis f. sp.tritici (Pgt) poses a significant threat to wheat production [2][3][4][5][6][7].It is found in most regions of the world and causes severe damage to wheat crops [8].It is estimated that 66% of global wheat-planting areas are susceptible to wheat stem rust, risking severe losses [9][10][11][12][13].However, since the 1970s, there has been some control of the disease through the cultivation and promotion of resistant cultivars and the manipulation of resistance-associated genes [14][15][16][17].Nevertheless, sudden outbreaks can occur, and thus continuous monitoring remains essential [18].Major epidemics of wheat stem rust in China are associated with the emergence of new virulent races, the loss of resistance in key cultivars, and the widespread cultivation of susceptible lines [19].Therefore, the cultivation and breeding of resistant cultivars are the most economical and effective measures for preventing and controlling wheat stem rust.
Resistance to stem rust in wheat is significantly associated with the Sr31 gene.However, the Pgt race Ug99 has shown virulence against Sr31 [20].Ug99 collections in Kenya were designated as TTKS based on the North American nomenclature system and later as TTKSK after adding a fifth set of differentials [21].The emergence of Ug99 underscores the threat posed by stem rust to global wheat production.Ug99 was recently found to have virulence not only against Sr31 but also against several other resistance genes in wheat, including Sr24, Sr38, and Sr36, as a result of successive mutations [5,22].In the 21st century, wheat stem rust, which had been absent from Europe for many years, reappeared.Between 2013 and 2014, TKTTF was detected in Germany, the United Kingdom, and Denmark.In 2016, thousands of hectares of durum wheat in Sicily, Italy, were affected by stem rust, the first significant outbreak of the disease in the region for 50 years [23].In 2020, wheat stem rust was detected in various regions of Switzerland, with transcriptomic analysis revealing a close genetic relationship between this strain and TKTTF [24].With the spread of Ug99 and its variants in African countries and the emergence of TKTTF and TTTTF in Europe, wheat stem rust has once again become a major concern [23][24][25].
The Huanghuai region of China is the country's largest wheat-producing area and has played a significant role in the epidemiology of wheat stem rust in China.Understanding the resistance of local cultivars (lines) and the expression of their disease-resistance genes is crucial [26].This study aimed to evaluate the resistance of 64 wheat cultivars collected from the southern part of the Huanghuai region, China, using two major Pgt races, 21C3CTHQM and 34MKGQM [25].Additionally, the molecular markers associated with the Sr genes Sr31, Sr24, Sr25, Sr26, and Sr38 were measured to assess the presence of these resistance genes.The research findings provide a reference for the prevention and control of wheat stem rust and for the breeding of resistant cultivars.

Field Evaluation
Field assessments of resistance were conducted during 2023 and 2024, evaluating the infection rates (IRs) of 64 wheat accessions with two major Pgt races, 34MKGQM and 21C3CTHQM (Table 1).Resistance identification usually begins at the end of May, when environmental temperature and humidity (Supplemental Table S1) are in the optimal period for the occurrence and spread of wheat rust.The results of the identification can accurately reflect the resistance of the wheat accessions to the tested races of Pgt.The results indicated that in 2023, five accessions were immune to 21C3CTHQM and 34MKGQM; 35 and 39 accessions were resistant against each of the races, respectively; and 24 and 20 accessions, respectively, were classified as susceptible (Table 2).In 2024, 12 and 14 accessions were immune to races 21C3CTHQM and 34MKGQM, respectively, while 29 and 30 accessions displayed resistance, with 23 and 20 accessions were classified as susceptible, respectively.Both races exhibited strong virulence against the evaluated cultivars.Notably, only two cultivars, Xinong 816 and Yimai 211, retained immunity over the two years, while the Xuyan 6, Xinong 1522, and Daimai 519 cultivars showed consistently high resistance during this period.In contrast, seven cultivars (Zhongzhimai 23, Longxing 1, Yunong 937, Huaguan 301, Wanke 800, Shaanhe 285, and Yunong 612) showed significant susceptibility over the two years.Nanpinzhongmai 6321 a Infection responses: severity/infection type/incidence; severity and incidence rates in the table are percentages; R: resistant, MR: moderately resistant, MS: moderately susceptible, S: susceptible, 0: immune.b "+" indicates the presence of detected genes, while "-" indicates the absence of detected genes.

Discussion
Sr24, which originates from Thinopyrum ponticum, is a primary resistance gene against stem rust in wheat cultivars grown in South Africa, Kenya, Australia, the United States, Europe, and the International Maize and Wheat Improvement Center (CIMMYT) in Mexico.The continuous mutation of Ug99 has led to the emergence of the new races TTKSK and TTKSP, which can overcome the resistance provided by Sr24 and thus pose a significant threat to wheat production [27].While some Pgt races exhibit virulence against Sr24, wheat cultivars carrying this gene still show good resistance to the new races TKTTF and TTRTF [28].In this study, a specific 500 bp fragment was amplified using Sr24#12 primers in a positive control.However, this fragment was not detected in the 64 wheat cultivars (lines) tested, indicating that Sr24 was not present in these cultivars [29][30][31][32].Despite its common use in wheat resistance breeding in other parts of the world, this gene appears to be relatively rare among wheat cultivars in China.Previous molecular testing of 449 wheat cultivars from Henan, Shandong, Shaanxi, Gansu, Xinjiang, Heilongjiang, Inner Mongolia, and Yunnan revealed that only one cultivar, Kenong 1006, contained Sr24.Given the strong resistance of Sr24 to Ug99, it is recommended that other resistance genes be aggregated and introduced into Chinese wheat cultivars during breeding efforts to enhance the genetic diversity of resistance traits in domestic cultivars.
The Sr25 and Sr26 genes are both known to provide excellent resistance to Ug99 and its variants, TKTTF and TTTRF, as well as to all races of Pgt found in China [31].In recent years, these two genes have been used extensively in wheat resistance breeding throughout the world.However, Sr25 is temperature-sensitive and provides reduced resistance in adult plants compared to seedlings, with raised temperatures significantly increasing the risk of susceptibility.Sr26 is commonly employed in wheat breeding programs in Australia [32].Based on previous molecular analyses and the findings of the present study, it appears that Chinese wheat cultivars generally lack both Sr25 and Sr26.Therefore, to enhance resistance to the new Ug99 and its variants, it would be advisable

Discussion
Sr24, which originates from Thinopyrum ponticum, is a primary resistance gene against stem rust in wheat cultivars grown in South Africa, Kenya, Australia, the United States, Europe, and the International Maize and Wheat Improvement Center (CIMMYT) in Mexico.The continuous mutation of Ug99 has led to the emergence of the new races TTKSK and TTKSP, which can overcome the resistance provided by Sr24 and thus pose a significant threat to wheat production [27].While some Pgt races exhibit virulence against Sr24, wheat cultivars carrying this gene still show good resistance to the new races TKTTF and TTRTF [28].In this study, a specific 500 bp fragment was amplified using Sr24#12 primers in a positive control.However, this fragment was not detected in the 64 wheat cultivars (lines) tested, indicating that Sr24 was not present in these cultivars [29][30][31][32].Despite its common use in wheat resistance breeding in other parts of the world, this gene appears to be relatively rare among wheat cultivars in China.Previous molecular testing of 449 wheat cultivars from Henan, Shandong, Shaanxi, Gansu, Xinjiang, Heilongjiang, Inner Mongolia, and Yunnan revealed that only one cultivar, Kenong 1006, contained Sr24.Given the strong resistance of Sr24 to Ug99, it is recommended that other resistance genes be aggregated and introduced into Chinese wheat cultivars during breeding efforts to enhance the genetic diversity of resistance traits in domestic cultivars.
The Sr25 and Sr26 genes are both known to provide excellent resistance to Ug99 and its variants, TKTTF and TTTRF, as well as to all races of Pgt found in China [31].In recent years, these two genes have been used extensively in wheat resistance breeding throughout the world.However, Sr25 is temperature-sensitive and provides reduced resistance in adult plants compared to seedlings, with raised temperatures significantly increasing the risk of susceptibility.Sr26 is commonly employed in wheat breeding programs in Australia [32].Based on previous molecular analyses and the findings of the present study, it appears that Chinese wheat cultivars generally lack both Sr25 and Sr26.Therefore, to enhance resistance to the new Ug99 and its variants, it would be advisable to introduce these genes into domestic wheat via resistance breeding to expand the genetic pool of resistance traits in Chinese wheat cultivars.
The Sr31 gene, located on the 1BL/1RS chromosome of wheat and transferred from rye "Petkus", has been widely used in wheat breeding since its introduction in China during the 1950s.It is estimated that nearly 50% of wheat cultivars may contain this gene.Although Sr31 has lost its resistance to the Ug99 race, it still maintains good resistance to the other races of Pgt found in China [29].The present study found that 30 of the 64 tested wheat cultivars (lines) from this region contained this gene, indicating a significant presence of Sr31 among Chinese cultivars.To date, no new races capable of overcoming Sr31-mediated resistance have been found in China [30][31][32].Consequently, cultivars carrying this gene have played a vital role in maintaining the stable population structure of Pgt in the country.Despite the loss of resistance to Ug99, Sr31 should still be judiciously employed in breeding programs for the effective prevention and control of stem rust recurrence in China.
The Sr38 gene, originating from Aegilops ventricosa, is associated with the leaf rustresistance gene Lr37 and the stripe rust resistance gene Yr17 [33].It provides robust resistance against three types of rust in wheat and is therefore widely utilized in wheat rust-resistance breeding worldwide.Consequently, Sr38 holds significant potential for future research on the prevention and control of wheat stem rust.Among the 64 wheat cultivars tested in this study, six were found to contain the Sr38 gene.Given the crucial role of Sr38 in preventing and controlling wheat stem rust in other parts of the world, as well as its marked resistance to wheat stem rust in China, these six cultivars could be used strategically with cultivars containing other resistance genes to enhance resistance in accordance with specific environmental conditions.
The present study evaluated the resistance of wheat cultivars from the southern part of the Huanghuai region to two Pgt races during 2023 and 2024.It was found that the resistance to the Pgt races 21C3CTHQM and 34MKGQM was poor.Of the 64 wheat cultivars tested, only Yimai 211 and Xinong 816 demonstrated immunity to both races during the 2023-2024 period.Among the tested materials, 20% of the accessions showed moderate susceptibility to susceptibility to the tested races, and some susceptible accessions had similar resistance levels to the tested race as the control variety LC, which had no resistance genes.The field severity and prevalence rates were as high as 100% (100S100).These plants began to dry up around 21-23 days after inoculation and had almost no yield at harvest.Molecular testing confirmed that 30 wheat cultivars (46.9%) contained the Sr31 resistance gene, indicating the significant presence of this gene among Chinese cultivars.Additionally, six cultivars were observed to contain the Sr38 gene.To date, no new races have been found to be capable of overcoming the resistance conferred by Sr31 and Sr38.Therefore, cultivars harboring these genes have been instrumental in maintaining the stable population structure of Pgt in China.Furthermore, wheat cultivars such as Daimai 519, Xinong 1522, Yannong 5066, and Zhengmai 6166, which exhibit resistance to races 21C3CTHQM and 34MKGQM, may possess additional genes conferring resistance to these two races.

Plant Materials and Pgt Races
The 64 wheat cultivars were provided by Associate Researcher Cao Tingjie from the Henan Academy of Agricultural Sciences.The races 21C3CTHQM and 34MKGQM were isolated, identified, and preserved at the Plant Immunology Laboratory of Shenyang Agricultural University for this study (Table 3).The susceptible variety Little Club (LC) and the single-gene, positive control lines LcSr24Ag (Sr24), Agatha/9*LMPG (Sr25), Eagle (Sr26), Sr31/6*LMPG (Sr31), and Trident (Sr38) for molecular detection were also provided by the Plant Immunology Laboratory of Shenyang Agricultural University.The field test for identifying resistance in adult plants was set up during spring in the experimental wheat field of the Plant Protection College of Shenyang Agricultural University.All winter wheat underwent vernalization treatment prior to sowing.Specifically, the tested wheat cultivars were placed in a culture dish and covered with a double layer of filter paper to induce germination.After the seedlings reached a height of about 1 cm, they were refrigerated at 4 • C for approximately 20 days of low-temperature vernalization and were subsequently sown in a single row 1 m in length with a row spacing of 25 cm.The LC variety was planted on the periphery as an inducing and protective row.
During the bolting stage, the wheat was inoculated (inoculation dates: 28 May 2023, 27 May 2024) using the powder-spraying method, ensuring that the soil remained completely moist during inoculation, which was carried out at sunset.Before inoculation, the plants were wetted with an aqueous solution of 0.05% Tween 20.The stem rust spores were then mixed with talcum powder in a 1:30 ratio and sprayed onto the wheat plants.After inoculation, the plants were covered with plastic film and kept moist for 16 h.
The plants were inspected once every five days for a total of three inspections, after which the LC cultivar used to check susceptibility was fully diseased.The resistance and susceptibility levels were classified according to the standards for wheat stem rust response types (Table 4).The IR classification included I (immune), NI (nearly immune), R (very resistant), MR (moderately resistant), MS (moderately susceptible), and S (susceptible).Disease severity was assessed according to the percentage of the surveyed leaf area occupied by uredinia, using benchmarks of 0.37% of the leaf area occupied by uredinia to represent a severity of 1% and 37% of the leaf area covered by uredinia to indicate a severity of 100%.Disease severity was classified into 12 levels, namely, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%.The climatic conditions are shown in Supplemental Table S1.

Molecular Markers of Resistance Genes
DNA was extracted from wheat using the CTAB method, and its quality was assessed by 1% agarose gel electrophoresis.Molecular markers closely linked to the resistance genes Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the presence of these genes in the 64 cultivars from the Huanghuai region.The primers used were synthesized by Sangon (https://www.sangon.com).The PCR amplification reaction system included 5 µL of Taq polymerase, 0.5 µL each of positive and negative primers, 1 µL of DNA, and ddH 2 O to make up 10 µL.The specific reaction conditions are detailed in Table 5.

Conclusions
This study evaluated the resistance of the adult plants of 64 wheat cultivars (lines) grown in the southern part of the Huanghuai region in China to two physiological races of Pgt.After the artificial inoculation of adult plants, it was found that 37 cultivars were resistant to both races 21C3CTHQM and 34MKGQM, while the remaining cultivars exhibited varying degrees of susceptibility.Molecular testing revealed that among these 37 resistant wheat cultivars, 32 contained either Sr31 or Sr38, while five did not possess any of the tested resistance genes, suggesting that their resistance mechanisms require further investigation.
Notably, cultivars such as Nanpin Yongminmai 2, Juliang 1208, Yimai 211, Zhengmai 6166, Xuyan 6, Bainongchengzhu 21, Xinong 1522, Xinong 816, and Tianyike 18 demonstrated high resistance to both races, showing immunity to near immunity in field conditions.These cultivars could be utilized selectively in the breeding process.Due to the infrequent global occurrence of wheat stem rust, resistance to this disease has not been a primary breeding goal in many regions, leading to a shortage of cultivars with strong resistance.With the continuous emergence and spread of new races of Pgt worldwide, the disease is poised to become a significant concern once again, making the exploration of resistance resources critical.
This study analyzed the resistance and conducted molecular testing on 64 wheat cultivars in the southern region of Huanghuai, preliminarily clarifying their resistance to current stem rust races and the distribution and frequency of resistance genes.The findings provide a theoretical basis for wheat resistance breeding and the strategic deployment of resistance genes.However, given the limited number of tests currently available, many known stem rust resistance genes were not evaluated.Therefore, it is necessary to further enhance the detection of resistance genes in cultivars to facilitate rational planting and breeding.

Table 1 .
Wheat accessions showing resistance and susceptibility at the adult plant stage to the two Pgt races during 2023 and 2024.

Table 2 .
Assessment of the resistance of tested wheat accessions to two races of Pgt at the adult plant stage.
a 5(7.8):The number outside the parentheses represents the number of cultivars, and the number inside the parentheses represents the percentage of that number.

Table 4 .
Host response and infection type descriptions used in wheat stem rust systems.

Table 5 .
Primers linked to rust resistance genes and PCR conditions.
: Weather conditions in Shenyang from 26 May to 26 June, 2023 and 2024.